Local direct thrombin inhibition preserves renal function by mitigating vascular damage and modulating pathophysiologic processes after onset of acute kidney injury

Abstract

Objective : To determine if thrombin-inhibiting nanoparticles preserve renal function when administered after onset of ischemia-reperfusion acute kidney injury (AKI), and to determine physiologic mechanisms by which this occurs. Hypothesis AKI therapy remains mainly supportive, as recent clinical trials of promising therapeutic agents have failed to preserve renal function. It has been previously shown that delivery of direct thrombin inhibiting nanoparticles prior to onset of AKI in mice has successfully preserved renal function. We hypothesized that inhibition of thrombin clotting and inflammatory signaling with the use of a sustained locally acting nanoparticle formulation might both preserve renal vasculature and broadly modulate pathophysiologic processes that contribute to renal ischemia reperfusion injury even after its onset. Methods C57BL/6 mice underwent bilateral warm ischemia for 17 minutes at 37°C followed by reperfusion. Two hours after reperfusion, mice were injected with either direct thrombin-inhibiting (n=10) or control (n=12) perfluorocarbon nanoparticles (PFC NP). The treatment PFC NP contained ~ 13650 copies per nanoparticle of the direct thrombin inhibitor, PPACK (Proline-Phenylalanine-Arginine-Chloromethyl-Ketone). After 24 hours, blood and kidneys were collected for evaluation of BUN, inflammatory eicosanoids and cytokines, histological analysis of vascular damage, endothelial activation, and cellular infiltrates. Results : Mice treated after AKI with direct thrombin-inhibiting PPACK NP exhibited significantly improved kidney function at 24 hours versus the control group (BUN: 62.10±8.71mg/dL vs 110.96 ±6.21mg/dL, p=0.0002). Vascular integrity was preserved as evidenced by increased expression of CD31 in both cortex (4.28±0.62% vs 1.75±0.34%, p=0.0018) and medulla (3.24±0.38% vs 1.91±0.29%; p=0.0073). Additionally, the PPACK NP group demonstrated significantly less acute tubular necrosis (ATN), further mitigating vascular damage (ATN: 24.44±1.76% vs 37.00±4.96%, p=0.035). Treatment reduced red blood cell trapping and subsequent iron accumulation in both cortex (3.37±0.50% vs 5.34±0.42%, p=0.0083) and medulla (0.50%±0.10% vs 1.01%±0.12%, p=0.006). Inflammatory markers were also reduced in the PPACK NP group, including endothelial VCAM-1 (positive area in medulla 0.49±0.12% vs 1.41±0.60%, p=0.0073), ICAM-1 (positive area in medulla 3.07±0.51% vs 4.46±0.35%, p=0.030). and serum M-CSF and IL-6. Mast cell infiltration and prostaglandin D2 (PGD2) were also decreased. Conclusion Nanoparticle delivery of local thrombin inhibitors that have a known minimal effect on systemic clotting parameters successfully protects renal function by preserving renal vasculature, reducing ATN and iron accumulation, and broadly attenuating thrombin-induced inflammatory mechanisms even when delivered after onset of AKI.